1. Field of the Invention
This invention relates to a method for immunochemical measurement of a trace component(s) using a spectral sensitizer as a photographically active substance.
2. Development of the Invention
Several methods for measuring a trace component(s) utilizing the combination of a photographically active substance and silver halide have been recently proposed by the present inventors. For example, methods in which a spectral sensitizer is employed as the photographically active substance are as follows:
(I) A method for immunologically measuring trace components which comprises: PA1 (II) A method for analyzing the location or distribution of a corresponding antibody or antigen or its receptor in tissue, utilizing the same dye-labelled antigen or antibody as used in (I) above, in combination with silver halide, which is subjected to a specific immune reaction. PA1 competitively reacting an antigen or antibody labelled with a spectral sensitizer and an antigen or antibody to be measured with an antibody or antigen which specifically reacts with the respective antigen species or antibody species, PA1 bringing either the thus formed reaction product or the unreacted component into contact with a silver halide light sensitive material, PA1 exposing the same to light having a wavelength which the spectral sensitizer absorbs, PA1 developing the exposed silver, and, PA1 quantitatively determining the antigen or antibody based upon the optical density of the resulting silver image and/or the color density obtained. PA1 (1) Straight, branched and cyclic alkyl groups, preferably containing 1 to 20 carbon atoms, such as a methyl group, an ethyl group, an isopropyl group, an n-dodecyl group, and a cyclohexyl group; PA1 (2) Aralkyl groups, preferably monocyclic and bicyclic aralkyl groups having an alkyl moiety containing 1 to 3 carbon atoms, such as a benzyl group; PA1 (3) Alkoxy groups, preferably containing 1 to 20 carbon atoms, such as a methoxy group and an ethoxy group; PA1 (4) Amino groups, preferably an --NH.sub.2 group and those amino groups mono- or di-substituted by an alkyl group containing 1 to 20 carbon atoms, such as a dimethylamino group and a diethylamino group; PA1 (5) Aryloxy groups, preferably a phenoxy group; PA1 (6) Groups represented by Ah--Xh--(Y).sub.nh -- PA1 (7) groups represented by ##STR2## (8) Groups represented by R.sup.3h CONHNH--Ar--Y"--. PA1 (a) Xh is a divalent linking group selected from the following x.sub.1 to x.sub.11 : x.sub.1 =--CSNH--, x.sub.2 =--S--CSNH--, ##STR3## x.sub.4 =--CONH--, x.sub.5 =--O--E--CONH--, ##STR4## x.sub.7 =--NHCO--, x.sub.8 =--O--, x.sub.9 =--SO.sub.2 NH--, x.sub.10 =--E--NH--, and x.sub.11 =--E=N--; PA1 (b) Y is a divalent linking group selected from the following y.sub.1 to y.sub.11 : y.sub.1 =--CONH--, y.sub.2 =--E--CONH--, y.sub.3 =--E--, y.sub.4 =--E--O--E'--, y.sub.5 =--E--S--E'--, y.sub.6 =--SO.sub.2 NH--, y.sub.7 =--E--SO.sub.2 NH--, y.sub.8 =--NHCONH--, y.sub.9 =--E--NHCONH--, y.sub.10 =--E--O--E'--CONH', and y.sub.11 =--E--E'--, PA1 wherein R.sub.11 is a hydrogen atom, an aliphatic group (preferably, an alkyl group containing 1 to 20 carbon atoms, a cycloalkyl group containing 3 to 12 carbon atoms, or an alkenyl group containing 2 to 20 carbon atoms), or an aromatic group (preferably, a phenyl group and a naphthyl group), PA1 R.sub.12 is a hydrogen atom or an aliphatic group represented by R.sub.11, PA1 R.sub.11 and R.sub.12 may combine with each other to form a ring, with preferred examples of such ring being ##STR5## (in this case, Ah represents hydrogen), when R.sub.11 and R.sub.12 do not form a ring, any one of R.sub.11 and R.sub.12 is a hydrogen atom, and PA1 E and E' each represents a saturated or unsaturated divalent aliphatic group (e.g., an alkylene group, such as an ethylene group and a 1-methylpropylene group, and an alkenylene group, such as propenylene group and a butenylene group), a divalent aromatic group (e.g., a phenylene group, a naphthylene group and a 5-amino-1,2-phenylene group), with the exception that in y.sub.11 =--E--E'--, E and E' are divalent groups different from each other and in x.sub.11 =--E=N--, E is --(CH.sub.2).sub.mh --CH.dbd. (wherein mh is an integer of 0 to 2); PA1 (c) nh is an integer of 0 or 1, and when nh is 1, particularly preferred combinations of Xh and Y are x.sub.3 -y.sub.2, x.sub.7 -y.sub.2, x.sub.8 -y.sub.2, x.sub.12 -y.sub.3, x.sub.3 -y.sub.7, x.sub.5 -y.sub.9, x.sub.9 -y.sub.9, and x.sub.3 -y.sub.10 ; and PA1 (d) Ah represents a straight, branched or cyclic alkyl group (preferably containing 1 to 20 carbon atoms, such as a methyl group, a propyl group, and an n-hexyl group), a monocyclic or bicyclic aryl group (e.g., a phenyl group), a monocyclic or bicyclic aralkyl group (preferably containing 7 to 26 carbon atoms, such as a benzyl group), and a heterocyclic radical. PA1 an alkoxy group (preferably containing 1 to 18 carbon atoms, such as a methoxy group), PA1 an alkoxycarbonyl group (preferably containing 2 to 19 carbon atoms, such as an ethoxycarbonyl group), PA1 a monocyclic or bicyclic aryl group (e.g., a phenyl group), PA1 an alkyl group (preferably containing 1 to 20 carbon atoms, such as a methyl group and a tert-amyl group), PA1 a dialkylamino group (preferably containing 1 to 20 carbon atoms, such as a dimethylamino group), PA1 an alkylthio group (preferably containing 1 to 20 carbon atoms, such as a methylthio group), PA1 a mercapto group, a hydroxy group, a halogen atom, a carboxy group, a nitro group, a cyano group, PA1 a sulfonyl group (preferably containing 1 to 20 carbon atoms, such as a methylsulfonyl group), and PA1 a carbamoyl group (preferably containing 1 to 20 carbon atoms, such as a carbamoyl group and a dimethylcarbamoyl group). PA1 (b) Bh is a hydrogen atom or a saturated or unsaturated aliphatic group [such as an alkyl group (preferably containing 1 to 20 carbon atoms, e.g., a methyl group and an ethyl group), an alkenyl group (preferably containing 2 to 22 carbon atoms, e.g., an allyl group), and an alkynyl group (preferably containing 2 to 20 carbon atoms, e.g., a butynyl group)], which may be substituted by an alkoxy group, an alkylthio group, an acylamino group, an acyloxy group, a mercapto group, a sulfo group, a carboxy group, a hydroxy group, a halogen atom, an amino group, or the like; PA1 (c) Y' has the same meanings as described for Y in Group (6); and PA1 (d) nh is 0 or 1. PA1 (a) R.sup.3h is the same as R.sup.2h as described hereinafter; PA1 (b) --Ar-- represents a divalent aryl group, preferably a phenylene group, which may be substituted; and PA1 (c) Y" is the same as Y described in Group (6), with divalent linking groups represented by y.sub.3 to y.sub.5 being particularly preferred. PA1 R.sub.3m and R.sub.4m have the same meanings as R.sub.c and R.sub.1c ; R.sub.5m has the same meaning as R.sub.2c ; r has the same meaning as n; L.sub.1 and L.sub.2 are as defined above: PA1 m.sub.1 represents 2, 3 or 4; PA1 d represents 1, 2 or 3; PA1 Q.sub.1m represents an oxygen atom, a sulfur atom or --N--R.sub.6m (R.sub.6m represents an aliphatic group); PA1 Q.sub.m represents a non-metallic atomic group necessary for completing a 5-membered or 6-membered nitrogen-containing heterocyclic nucleus; examples of such heterocyclic nuclei include a 2-pyrazolin-5-one nucleus (e.g., 3-methyl-1-phenyl-2-pyrazolin-5-one nucleus, etc.), an isoxazole nucleus, an oxyindole nucleus, a barbituric acid nucleus or 2-thiobarbituric acid nucleus, a rhodanine nucleus (e.g., rhodanine nucleus, 3-sulfoalkyl-rhodanine nuclei, 3-sulfoaryl-rhodanine nuclei, 3-alkyl-rhodanine nuclei, etc.), a 2,4-thiazolidinone nucleus, a thiazolidinone nucleus, a 2,4-imidazolidinedione (hydantoin)nucleus, a 2-thio-2,4-imidazolidinedione nucleus, etc. PA1 L.sub.1 and L.sub.5 are the same as L.sub.1 to L.sub.3 ; PA1 R.sub.10r is the same as R.sub.4m ; Q.sub.2r is the same as Q.sub.1m ; PA1 k and l represent 1, 2 or 3, and may be the same or different. PA1 (1) Spectral sensitizers are directly reacted with the aforesaid functional groups; PA1 (2) Spectral sensitizers and the aforesaid functional groups are reacted using an activating agent, and PA1 (3) Spectral sensitizers and the aforesaid functional groups are reacted through at least one compound having a bifunctional group.
competitively reacting an antigen or antibody labelled with a spectral sensitizer and an antigen or antibody to be measured with an antibody or antigen which specifically reacts with the antigen or antibody, PA2 bringing either the thus formed dye-labelled antigen-antibody reaction product or the unreacted antigen or antibody into contact with silver halide, PA2 exposing the same to a light having spectrally sensitizing wavelength corresponding to the spectral sensitizer, PA2 developing the exposed silver halide, and, PA2 measuring the resulting optical density of the developed silver and/or colored dye; and,
Radioimmunoassay (hereafter merely "RIA") is a method for the assay of a trace component(s) utilizing a specific antigen-antibody reaction. The basic principles of RIA are as follows. The reaction of a substance labelled or marked with a radioactive isotope (RI) in a given amount and a substance having a specific binding affinity thereto in a given amount results in a coupled product of both of these components, while a part of the labelled substance remains in an unbound or unreacted free state. The reaction proceeds based on the laws of mass action in general, and, therefore, when an unlabelled substance is added to the reaction system, binding with a limited amount of binding protein is decreased and a certain relationship (calibration curve) can be established therebetween. As a result, an amount of an unknown substance can be determined from the calibration curve if the bound substance and the labelled substance in the free state are separated and either one or both are measured with respect to the RI amount.
Due to the high sensitivity and the simplicity of RIA, RIA is particularly applicable to the measurement and inspection of trace amounts of proteins in blood and hormones. Details thereon are given in, e.g., Kumahara and Shizume, NEW RADIOIMMUNOASSAY, pages 3 to 10 (1977), published by Asakura Publishing Co., Ltd., Tokyo, KISO SEIKAGAKU JIKKENHO (Basic Biochemical Experiments) (6), subtitled "Biochemical Assay" (1967), published by Maruzen Co., Ltd., Tokyo, P. D. Boyer et al, The Enzyme, vols. 3, 4 and 5 (1971), published by Academic Press, New York and, METHODS IN ENZYMOLOGY, edited by Sidney P. Colowick et al, vols. I, II, III, V and VII, published by Academic Press, New York.
However, RIA is subject to several disadvantages due to the use of RI labelling substances (.sup.125 I, .sup.131 I, etc.) which must have high specific radioactivity to maintain immune activity and must be of high purity. For these reasons, RIA involves the danger of radiation exposure and it is necessary to use expensive and unstable labelling substances which cannot be used for extended periods of time. In addition, special installations, equipment and personnel qualified to deal with radiation are required. Finally, after RIA, disposal of radioactive waste material and the ensuing pollution problems are encountered.
For these reasons, it has been desired to develop a method for the immunological measurement of trace components which is stable and provides sufficient sensitivity without using any isotope.
The inventors have already proposed a method for the measurement of trace components with high sensitivity applicable to immunological measurement using a photographically active substance, e.g., a spectral sensitizer, in combination with silver halide. Such an immunological measurement method previously proposed by the present inventors is basically practiced as follows.
The immunological measurement method comprises:
More specifically, a known amount of a labelled antigen or labelled antibody is reacted with an antigen or antibody. After separating the reaction product and unreacted material, a quantitative assay of the labelling substance (either one) is performed using silver halide to prepare a calibration curve. Based on the calibration curve, an unknown amount of antigen or antibody can be determined. This is because the optical density of the thus formed blackened areas and/or colored dye is proportional to the amount of the spectral sensitizer adsorbed on silver halide, which is in turn proportional to the amount of the antigen or antibody to be measured.
Based upon such a new principle, the present inventors have discovered a basic measurement method for an antigen or antibody as described above (also see U.S. Ser. No. 126,920 filed Mar. 3, 1980 now U.S. Pat. No. 4,337,063, which is hereby incorporated by reference). While this novel photochemical measurement of an antigen or antibody eliminates problems accompanied by RIA and enzyme immunoassay, it is still desired to develop a measurement method having a more improved detection sensitivity in order to obtain results in good reproducibility and high accuracy even in the case where an antigen or antibody to be measured is present in a minute quantity as in the order of .mu.g/ml or less, or even in the case where the immune activity of an antigen or antibody to be measured is very weak.
The present inventors have now discovered that detection sensitivity with silver halide is markedly enhanced when a specific hydrazine compound is present at any stage from bringing the substances labelled with the spectral sensitizer into contact with silver halide to developing silver halide.